One component fibrin glue comprising zymogens

ABSTRACT

Provided herein is a single component sealant formulation (e.g. in a liquid form), methods for its preparation, and use. The formulation includes fibrinogen; vitamin K-dependent clotting zymogens comprising at least Factor II (FII) and Factor X (FX).

FIELD OF THE INVENTION

Provided herein is a single component sealant formulation e.g. in aliquid form, methods for its preparation, and methods of use thereof.

BACKGROUND

Fibrin sealants, also known as fibrin glue, have been in use in theclinic for decades (see, for example, Tabélé, et al. J Pharm PharmaceutSci 2012, 15:124-140; Dickneite, G et al. Thrombosis Res 2003,112:73-82). Oftentimes, fibrin sealant consist of two liquid components,a fibrinogen comprising component and a thrombin comprising component,which are stored frozen due to their inherent instability. Sometimesfibrin sealant products consist of two freeze dried components, whichrequire reconstitution immediately prior to use and delivery by aconjoined syringe or other double-barreled delivery device. Freeze driedformulations are typically stable, but the fibrinogen component isdifficult to reconstitute.

A fibrin sealant clot is formed by enzymatic reactions involvingfibrinogen, thrombin and Factor XIII. The thrombin converts thefibrinogen to fibrin by enzymatic action at a rate determined by theconcentration of thrombin. Factor XIII, an enzyme of the bloodcoagulation system, cross-links and stabilizes the fibrin clot. Thisprocess bypasses most of the steps of normal coagulation and mimics itslast phase. Some manufacturers add anti-proteolytic agents to the fibringlue formulation (e.g. as described in WO93/05822) or specificallyremove the plasminogen in order to stop or delay fibrinolysis (e.g. asdescribed in U.S. Pat. Nos. 5,792,835 and 7,125,569).

The thrombin component contains the enzyme thrombin, which is a serineprotease, and can be from human or animal (e.g. bovine or porcine)origin or recombinantly produced. The fibrinogen component can be fromhuman or animal origin or recombinantly produced. Upon mixing thetwo-component solutions, thrombin cleaves fibrinogen thus allowing thelatter to generate fibrin polymers/sealant.

Thrombin displays high specificity toward fibrinogen and cleaves adefined sequence in the fibrinogen molecule, however, at very highconcentrations thrombin can undergo auto-proteolysis. Theauto-proteolytic properties of thrombin may result in reduced activityand instability of the thrombin component of fibrin sealant.

Background art includes U.S. Pat. Nos. 5,219,328; 5,318,524; 8,367,802;6,500,427; 5,750,657; 6,262,236; 6,268,483; and US Patent ApplicationPublication No. 2013/0149292.

SUMMARY OF THE INVENTION

Provided herein are single component, stable sealant formulations,methods of manufacture and methods of use, which eliminate thecumbersome steps involved in manufacturing, preparing and/or using theknown sealant formulations.

In one aspect, provided herein is a sealant formulation comprising aneffective amount of fibrinogen, vitamin K-dependent clotting zymogenscomprising at least Factor II (FII), and Factor X (FX), and at least onereversible inhibitor of at least one of the vitamin K-dependent clottingzymogen; wherein the formulation is free of an added irreversiblethrombin inhibitor.

In another aspect, provided herein is a calcium-free sealant formulationcomprising fibrinogen and vitamin K-dependent clotting zymogenscomprising at least Factor II and Factor X.

The term “effective amount” for fibrinogen, vitamin K-dependent clottingzymogens and the reversible inhibitor are such that, effectively, littleor no premature activation (e.g. coagulation, clotting and/or conversionfrom zymogen to an active enzyme) takes place, yet the formulationspontaneously coagulates and forms a sealant upon dilution,neutralization, blockage and/or removal of the reversible inhibitor. Forexample spontaneous coagulation and sealant formation may occur bycontact with a bleeding surface, following small molecule exchange,and/or addition of an activator e.g. free calcium.

In some embodiments, the formulation further comprises Factor V (FV).

In some embodiments, the vitamin K-dependent clotting zymogens furthercomprise Factor IX (FIX).

In some embodiments, the vitamin K-dependent clotting zymogens furthercomprise Factor VII (FVII).

In some embodiments, Factor X, Factor VII, and/or Factor IX are, atleast partially, in their active form.

In some embodiments, the formulation is in liquid form. In someembodiments, the formulation comprises a pharmaceutically acceptablecarrier. The liquid formulation exhibits extended stability and remainsstable for at least 14 days at an ambient temperature selected from thegroup consisting of about 2, 3, 4, 5, 6, 7, and 8° C. In someembodiments, the liquid formulation remains stable for about 30, 35, 45days, 60 days and up to 90 days or more, at a temperature of about 2° C.to 8° C. “Ambient temperature” is the temperature in the surroundingswhere the sealant formulation is kept.

In some embodiments, the liquid formulation is stable for about or atleast 7 days at an ambient temperature in a range of about 2° C. and upto room temperature.

In certain embodiments the liquid formulation is stable for about 30days at room temperature.

“Room temperature” typically refers to a temperature of about 20° C. to25° C.

The liquid formulation can be an aqueous liquid formulation.

Stability can be determined by observing minimal or absence ofspontaneous clotting in the formulation e.g. the formulation does notshow or have spontaneous clotting in the absence of an activator, suchas free calcium, and retains its clotting activity level upon exposureto calcium. The clotting activity level or capability of the formulationto form a sealant can be determined in-vitro and/or in-vivo. Stabilitycan also be determined by measuring and observing the presence ofminimal or absence of fibrin formation in the shelf-ready aqueousformulation.

Fibrin polymerization or clotting can be measured, for example, bymeasuring migration length on a slanted surface (or drop test model) orby any other method known in the art. Full polymerization can beassessed by cessation of flow of the liquid formulation upon inversion.Rapid polymerization can be measured using a Stat4 clotting analyzerStago Diagnostics or similar coagulometer.

The term “activator” refers to an agent that can initiate, facilitateand/or accelerate the conversion of a zymogen into an active enzyme. Theterm “activator” herein is interchangeable with the term “initiator”.

In one embodiment, the source of the vitamin K-dependent clottingzymogens is Prothrombin-Proconvertin-Stuart Factor-Antihemophilic FactorB (PPSB) and/or a three-factor or four-factor Prothrombin ComplexConcentrate (PCC).

In some embodiments, PPSB includes Factors II, VII, IX and X.

In some embodiments, PCC includes Factors II, IX and X (three-factorPCC) and may further include Factor VII (four-factor PCC).

In various embodiments, vitamin K-dependent clotting zymogens is aconcentrate of vitamin K-dependent clotting zymogens comprising at leastFII and FX, concentrated by about 2-50 fold compared to theconcentration of these zymogens in plasma, as normalized to Factor II.The concentrate may be concentrated about 5 to about 40 fold, about 10fold or about 20 fold.

In various embodiments, vitamin K-dependent clotting zymogens is a PPSBconcentrate, or concentrated vitamin K-dependent clotting zymogenscomprising at least FII, FIX and FX, concentrated by about 2-50 foldcompared to the concentration of these zymogens in plasma, as normalizedto Factor II. The PPSB concentrate may be concentrated about 5 to about40 fold, about 10 fold or about 20 fold.

In some embodiments, the fibrinogen is present in the formulation in aneffective amount of about 1 to 2 mg/ml, 1 to 110 mg/ml, 10 to 110 mg/mlsuch as about 40 mg/ml to 70 mg/ml.

In one embodiment, the ratio of the active components: fibrinogen,vitamin K-dependent clotting zymogens, and the reversible inhibitor ofat least one of the vitamin K-dependent clotting zymogens, are suchthat, effectively, little or no premature activation (e.g. coagulation,clotting and/or conversion from zymogen to an active enzyme) takesplace, yet the formulation spontaneously coagulates and forms a sealantupon dilution, removal of the reversible inhibitor, blockage and/orneutralize of the inhibitor e.g. by contact with a bleeding surfaceand/or following small molecule exchange and/or addition of an activatore.g. free calcium.

“A reversible inhibitor of a vitamin K-dependent clotting zymogen” is anagent which effectively prevents or reduces premature activation (e.g.coagulation, clotting and/or conversion from a zymogen to an activeenzyme).

In some embodiments, the formulation is free of added thrombin.Accordingly, there is no requirement for a thrombin inhibitor and theformulation is free of added irreversible thrombin inhibitor or isessentially free of an irreversible thrombin inhibitor. An irreversiblethrombin inhibitor comprises a group of molecules that bind thrombincovalently or with a very high affinity (e.g. at a picomolar level) e.g.hirudin, and/or destroy a functional group on thrombin or render thethrombin inactive. For example hirudin and antithrombin III areconsidered herein as examples of such irreversible thrombin inhibitors.In some embodiments, the formulation is free of hirudin. In oneembodiment, the formulation is free of antithrombin III.

The term “free of added” in connection with the terms “free of addedthrombin” and “free of added irreversible thrombin inhibitor” means thatthe formulation is not supplemented with thrombin or irreversiblethrombin inhibitor. However, it should be noted that the formulation maycomprise low amounts of thrombin (e.g. less than 1 IU/ml formulation)and/or irreversible thrombin inhibitor (e.g. less than 5 μM) originallypresent in the formulation and/or thrombin spontaneously formed in theformulation.

The formulation includes at least one reversible inhibitor of at leastone of the vitamin K-dependent clotting zymogens. Such inhibitor is anagent that can substantially prevent initiation and/or delays theconversion of a zymogen into an active enzyme. The inhibitor can beselected from heparin, a calcium chelator, a reversible serine proteaseactive site inhibitor and a combination of such inhibitors.

Typically, a reversible inhibitor relates to a low affinity inhibitorhaving no permanent effect on protein activity. Therefore, typicallydilution will remove the inhibitory effect.

At least one vitamin K-dependent clotting zymogen reversible inhibitormay be heparin. The vitamin K-dependent clotting zymogen reversibleinhibitor which inhibits generation of an active enzyme may be a calciumchelator, for example a citrate ion, EDTA, EGTA, oxalate or acombination of such calcium chelators.

In some embodiments, the calcium chelator is a citrate ion, for exampleprovided by sodium citrate. The formulation may include from about 1 mMto about 50 mM sodium citrate, or from about 5 mM to about 25 mM sodiumcitrate. In some embodiments, the calcium chelator is EDTA and/or EGTA.The formulation may include from about 0.1 mM to about 2.5 mM EDTAand/or EGTA. In some embodiments, the calcium chelator is oxalate. Insome embodiments, the formulation includes a combination of a citrateion, oxalate and EDTA and/or EGTA, for example EDTA and a citrate ionprovided by sodium citrate. In some embodiments, the formulationcomprises from about 0.1 mM to about 2.5 mM EDTA and/or EGTA.

In some embodiments, the at least one vitamin K-dependent clottingzymogen reversible inhibitor is a serine protease active site inhibitor,for example arginine, lysine, benzamidine or a combination of suchserine protease active site inhibitors. The formulation may include, forexample, arginine in an amount from about 0.1% to about 5% (w/v), about0.5% to about 4% (w/v) arginine, or about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%,or 4% (w/v) arginine.

In some embodiments, the ratio of vitamin K-dependent clotting zymogens(U), including PPSB, vitamin K-dependent clotting zymogens comprising atleast FII, FIX and FX, or PCC, to fibrinogen (mg clottable protein) isabout 0.01 to about 1.0, as normalized to Factor IX or Factor II. Insome embodiments, the ratio is about 0.05 to about 0.2, or about 0.1 toabout 0.2.

In some embodiments, the ratio of vitamin K-dependent clotting zymogens(U), including PPSB, vitamin K-dependent clotting zymogens comprising atleast FII, and FX, or PCC, to fibrinogen (mg clottable protein) is about0.01 to about 1.0, as normalized to Factor II. In some embodiments, theratio is about 0.05 to about 0.2, or about 0.1 to about 0.2.

The formulation is preferably sterile and free from pathogens, forexample by pasteurization and/or filtration.

The formulations disclosed hereinabove are useful in, for example,hemostasis, healing, and/or surgery, including, without limitation,graft fixation, wound healing and sealing of anastomosis sites. Theformulation can also be used in plastic surgery, for example,abdominoplasty; skin and internal organ graft fixation; tissue healing;burn treatment; and/or attenuating wound bleeding. Furthermore, theformulation is useful for dura sealing, for example in cranial or spinalsurgery.

Accordingly, in one aspect, provided is a method of providing hemostatictreatment; graft fixation, wound healing and/or anastomosis, to asurface in a subject, comprising applying to the surface a formulationaccording to the invention. The method includes, without limitations,abdominoplasty; tissue healing; burn treatment; and dura sealing. Thesubject may be a human subject.

In another aspect, provided is a formulation according to the inventionfor use in healing, hemostasis and/or surgery. The uses include, withoutlimitation, graft fixation; wound healing; anastomosis; abdominoplasty;tissue healing; burn treatment; and dura sealing.

In another aspect, provided herein is a method for preparing a sealantat a surface comprising:

a) providing a formulation according to the invention; and

b) applying the formulation to the surface under conditions whichfacilitate fibrin polymerization at the surface.

The surface can be a bleeding or non-bleeding surface in a subject. Thesurface may also be for example, a bodily surface, an external orinternal body organ, a blood vessel or a graft tissue or organ.

In some embodiments, the conditions described in the above methodinvolve applying the formulation directly to a bleeding or non-bleedingsurface in a subject.

Several conditions of activation are disclosed. For example, the mixtureof PPSB and fibrinogen may be applied directly onto a bleeding site inwhich case the inhibitors are diluted and the concentrated zymogens andfibrinogen function in order to rapidly bring about hemostasis.

In some embodiments, the conditions involve (i) removing, diluting,blocking and/or neutralize the reversible inhibitor of at least one ofthe vitamin K-dependent clotting zymogens and/or (ii) adding a smallmolecule activator of at least one of the vitamin K-dependent clottingzymogens.

The small molecule activator may be a phospholipid or a cation, forexample a calcium cation or other divalent cations such as magnesium,iron or zinc or combinations thereof. In some embodiments, the cation isa calcium cation, provided by CaCl₂.

In some embodiments, the small molecule activator is a phospholipid suchas phosphatidylserine, phosphatidylcholine, phosphatidylinositol orphosphatidylethanolamine

A small molecule activator or reversible inhibitor has a molecularweight of up to 1 kilodalton.

In various embodiments, the step of removing or diluting the inhibitorof at least one of the vitamin K-dependent clotting zymogens is carriedout by passing the formulation through a small molecule exchange devicee.g. a column prior to or during application to the surface of asubject.

Typically, small molecule exchange is the replacement of one set ofsmall molecules with another set. Oftentimes, the resin in the column ispre-equilibrated with the small molecules that are desired in the finalformulation and/or small molecules that facilitate and/or accelerate theconversion of a zymogen into an active enzyme. The resin beads aretypically porous, the pores being in the range of molecular weights ofthose molecules which are to be replaced. In one embodiment, a liquidformulation comprising the zymogens and fibrinogen is passed through acolumn that is packed with the porous resin. The zymogens and fibrinogenin the solution will be too large to enter the pores of the resin andwill quickly pass through the column. Without being bound by themechanism, small molecule in the solution or formulation e.g. thereversible inhibitors, will travel a more tortuous path, as they areable to enter and re-exit the pores of the resin, thus greatly slowingtheir rate of migration through the resin bed. Those small molecule withwhich the resin has been pre-equilibrated enjoy an advantage of asignificant head-start, and therefore exit the resin together with theproteins (the zymogens and fibrinogen). Thus, the buffer salts and othersmall molecules are exchanged in this step.

In some embodiments, the at least one vitamin K-dependent clottingzymogen inhibitor is diluted at the surface of bleeding or non-bleedingwound or an internal or external organ of a subject. The subject may bea human patient. The small molecule exchange device includes forexample, water, saline, CaCl₂ or other divalent cations, and/or aphospholipid.

In some embodiments, the step of adding the small molecule activator iscarried out by passing the formulation through a small molecule exchangedevice including the small molecule activator prior to or duringapplication to the surface.

In another embodiment, the small molecule is added manually. In someembodiments, the small molecule activator is selected from aphospholipid and a divalent cation, such as a calcium and iron cation.The small molecule exchange device may contain, for example, a calciumcation containing buffer.

In some embodiments of the method of treatment, therapeutic use and/ormethod of forming/preparing/manufacturing a sealant, the vitaminK-dependent clotting zymogens in the formulation comprise at leastFactor II, Factor X and may further comprise Factor VII and/or FactorIX. The source of the vitamin K-dependent clotting zymogens may be, forexample, Prothrombin-Proconvertin-Stuart Factor-Antihemophilic Factor B(PPSB) and/or Prothrombin Complex Concentrate (PCC). In someembodiments, the PPSB includes Factors II, VII, IX and X. The PCC mayinclude Factors II, and X and may optionally further include Factor IX,Factor V and/or Factor VII. In one embodiment, the vitamin K-dependentclotting zymogen source is a PPSB concentrate, concentrated by about2-50 fold compared to the zymogens concentration in plasma, asnormalized to Factor IX and/or Factor II. The PPSB concentrate may beconcentrated about 4 to 40 fold, about 5 to 40 fold, about 10 fold orabout 20 fold.

In some embodiments of the method of treatment, therapeutic use and/ormethod of forming/preparing/manufacturing a sealant, the formulationincludes a pharmaceutically acceptable carrier. In some embodiments, theformulation is a liquid formulation.

In preferred embodiments of the methods, the liquid formulation remainsstable for at least 14 days at an ambient temperature of about 2° C. to8° C. In some embodiments, the liquid formulation remain stable for atleast 30 days, 45 days, 60 days, up to 90 days or more, at an ambienttemperature of about 2° C. to 8° C.

In some embodiments, the liquid formulation is stable for about or atleast 7 days at an ambient temperature in a range of about 2° C. and upto room temperature.

In certain embodiments the liquid formulation is stable for about 30days at room temperature.

In some embodiments, the formulation is free of added thrombin and isessentially free of an irreversible thrombin inhibitor such as hirudin.Preferably, the formulation is free of added antithrombin III orsubstantially free of antithrombin III, an irreversible thrombininhibitor.

The inhibitor of at least one of the vitamin K-dependent clottingzymogens is selected from heparin (in the absence of added antithrombinIII), a calcium chelator, a reversible serine proteases active siteinhibitor and a combination of such inhibitors. The vitamin K-dependentclotting zymogens inhibitor may be heparin. The vitamin K-dependentclotting zymogens inhibitor may be a calcium chelator, for example acitrate ion, oxalate, EDTA, EGTA or a combination of such calciumchelators. In some embodiments, the calcium chelator is a citrate ion,for example provided by sodium citrate. The formulation may include fromabout 1 mM to about 50 mM sodium citrate, or from about 5 mM to about 25mM sodium citrate.

In some embodiments, the calcium chelator is EDTA and/or EGTA. Theformulation may include from about 0.1 mM to about 2.5 mM EDTA and/orEGTA. In some embodiments, the calcium chelator is oxalate. Theformulation may include a combination of a citrate ion and EDTA and/orEGTA, for example sodium citrate and EDTA.

In some embodiments of the method of treatment, therapeutic use and/ormethod of forming/preparing/manufacturing a sealant, the at least onereversible inhibitor of at least one vitamin K-dependent clottingzymogen in the formulation is a serine protease active site inhibitor,for example arginine, lysine, benzamidine or a combination of suchreversible serine protease active site inhibitors. The formulation mayinclude arginine in an amount of from about 0.1% to about 5% (w/v)arginine, from about 0.5% to about 4%, or about 1%, 1.5%, 2%, 2.5%, 3%,3.5%, or 4% arginine.

In some embodiments of the method of treatment, therapeutic use and/ormethod of forming/preparing/manufacturing a sealant, the ratio of PP SB,vitamin K-dependent clotting zymogens or PCC (U) to fibrinogen (mgclotable protein) in the formulation is about 0.01 to about 1.0, asnormalized to Factor IX and/or Factor II. In some embodiments, the ratiois about 0.05 to about 0.2, or about 0.1 to about 0.2.

Further provided is a container comprising a formulation disclosedherein. In some embodiments, the container is an ampoule, a vial, a testtube or a syringe and the like. The formulation may be liquid or solid.

In various embodiments, provided is a syringe or an applicatorcontaining the formulation of the invention that is attached to a smallmolecule exchange device. The device comprises a small moleculeactivator of a vitamin K-dependent clotting zymogen. The small moleculeactivator may be for example, a phospholipid, or a divalent cation, suchas a calcium cation and or iron cation. The small molecule exchangedevice may contain, for example, a calcium cation containing buffer.

In another aspect, provided is a kit comprising a container such as anampoule, a vial or syringe which includes the formulation as disclosedhereinabove; optionally the kit includes a small molecule exchangedevice and/or instructions for use. A kit may include at least onecontainer and at least one label. Suitable containers include, forexample, ampoules, vials, syringes and test tubes. The containers can bemade of for example, glass, metal or plastic.

In some embodiments, the small molecule exchange device is a gelfiltration column, for example a disposable desalting column of about0.5 to about 5 ml for use by gravity flow and/or centrifugation. Thedevice may include a solvent, for example water or saline, and/or mayinclude a small molecule activator of at least one of the vitaminK-dependent clotting zymogens, such molecule being for example CaCl₂.The device may also be any commercially available gel filtration deviceof any conformation.

Further disclosed herein is a method of manufacturing a sealantformulation according to the invention, the method includes the stepsof:

a) providing a fibrinogen component;

b) providing a component comprising the vitamin K-dependent clottingzymogens comprising at least FII, FX and optionally FIX and/or FVII;

c) providing a component comprising at least one reversible inhibitor ofat least one of the vitamin K-dependent clotting zymogens; and

d) admixing the components of a) to c); wherein the admixed componentsare free of added irreversible thrombin inhibitor.

The components can be provided in any combination, for example thefibrinogen component can be combined with the component comprising thereversible inhibitor; the fibrinogen component can be combined with thevitamin K-dependent clotting zymogens component; and/or the vitaminK-dependent clotting zymogens component can be combined with thereversible inhibitor.

The term “admixing” means mixing the components in any order, anycombination and/or sub-combination.

In some embodiments, at least one of the components comprises FV. Inanother embodiment, an additional component comprising FV is admixedwith components a) to c).

In some embodiments, at least one of the components is provided inliquid form, thereby resulting in a liquid formulation.

A liquid formulation can be dried by including a drying step such aslyophilization.

In one embodiment, the method of manufacture further comprises a dryingstep, thereby resulting in a dry formulation.

Further provided is a sealant formulation obtainable by the method ofmanufacturing a sealant formulation according to the invention.

In another aspect, the invention provides a sealant formulationcomprising fibrinogen; vitamin K-dependent clotting zymogens comprisingat least Factor II, Factor IX and Factor X; and at least one reversibleinhibitor of at least one of the vitamin K-dependent clotting zymogen,wherein the formulation is free of added irreversible thrombininhibitor. In another aspect, the invention provides a method forpreparing a sealant at a surface comprising: providing the formulationdisclosed hereinabove; and applying the formulation to the surface underconditions which facilitate fibrin polymerization at the surface.

In certain embodiments, the conditions which facilitate fibrinpolymerization comprise removing, neutralizing, blocking and/or dilutingthe reversible inhibitor of at least one of the vitamin K-dependentclotting zymogens by passing the formulation through a small moleculeexchange device prior to or during application to the surface of asubject. In some embodiments, the at least one vitamin K-dependentclotting zymogen inhibitor is diluted at the surface of bleeding ornon-bleeding wound or an internal or external organ of a subject. Thesubject may be a human patient. The small molecule exchange deviceincludes for example, water, saline, CaCl₂ or other divalent cationsand/or a phospholipid.

In various embodiments, the removing, blocking, neutralizing, ordiluting the inhibitor of at least one of the vitamin K-dependentclotting zymogens is carried out by applying the formulation directly toa surface of a body part of a subject. For example, the surface may be ablood vessel or bleeding tissue or organ.

In another aspect, the invention provides a calcium-free sealantformulation. Such formulation can be obtained by capturing all calciuminitially present in one of the components e.g. capturing the calcium byusing a chelator such as EDTA or EGTA, and removing the calcium-chelatorcomplex formed e.g. by using a size exclusion filter.

Accordingly, provided herein is a calcium-free sealant formulation e.g.in liquid form comprising fibrinogen; and vitamin K-dependent clottingzymogens comprising at least Factor II, Factor X, and optionally FactorIX.

The term “calcium-free” means that the formulation contains less than1.25 mmol/L. In one embodiment, the formulation is also free of addedirreversible thrombin inhibitor.

In some embodiments, such calcium-free formulation is free of achelating agent.

In some embodiments, the vitamin K-dependent clotting zymogens furthercomprise Factor VII.

In some embodiments, the vitamin K-dependent clotting zymogens are aPPSB, PPSB plasma fraction or PCC.

In some embodiments, the vitamin K-dependent clotting zymogens are aPPSB plasma fraction or PCC.

In some embodiments, the formulation is free of added thrombin.

In some embodiments, the irreversible thrombin inhibitor is selectedfrom the group consisting of hirudin, small molecule thrombininhibitors, and antithrombin III.

The reversible and irreversible inhibitors mentioned herein can besynthetic or natural.

The vitamin K-dependent clotting zymogens may be a concentrate,concentrated by about 2-50 fold compared to the vitamin K-dependentclotting zymogens concentration in plasma, as normalized to Factor IXand/or Factor II, for example a Prothrombin Complex Concentrate (PCC) ofPPSB. In some embodiments, the PCC is a three-factor PCC or afour-factor PCC. The ratio of PPSB or PCC (U) to fibrinogen (mgclottable protein) is about 0.01 to about 1.0 as normalized to Factor IXand/or Factor II.

The formulation may further comprise a reversible inhibitor of at leastone of the vitamin K-dependent clotting zymogens wherein the inhibitorof at least one of the vitamin K-dependent clotting zymogens is selectedfrom the group consisting of heparin (in the absence of addedantithrombin III), a serine protease active site inhibitor and acombination of such inhibitors of the vitamin K-dependent clottingzymogens.

In some embodiments, the reversible inhibitor of at least one of thevitamin K-dependent clotting zymogens is a serine protease active siteinhibitor. In some embodiments, the serine protease active siteinhibitor is selected from the group consisting of arginine, lysine,benzamidine and a combination of such serine protease active siteinhibitors. In one embodiment, the serine protease active site inhibitoris arginine.

The formulation may be used in hemostasis, sealing, tissue adhesion,graft fixation, wound healing or anastomosis.

Further provided is a method for preparing a sealant at a surfacecomprising: providing the liquid, calcium-free formulation and applyingthe formulation to the surface under conditions which facilitate fibrinpolymerization at the surface.

In some embodiments, the surface is a blood vessel or an internal orexternal body organ.

In some embodiments, the conditions comprise adding a small moleculeactivator of vitamin K-dependent clotting zymogens e.g. calcium cation,thereby causing fibrin polymerization.

In various embodiments, the addition of the small molecule activator iscarried out by passing the formulation through a small molecule exchangedevice prior to or during application to the surface of a subject. Thesmall molecule exchange device includes for example, water, saline,CaCl₂ or other divalent cation and/or a phospholipid.

The formulations according to the invention may be kept in a container,for example an ampoule, test tube, a vial or a syringe.

Further provided is a kit comprising the formulation or the containeraccording to the invention, a small molecule exchange device; andoptionally instructions for use.

Further provided is a method of manufacturing the calcium-free sealantformulation comprising the steps:

a) providing a fibrinogen component;

b) providing a component comprising vitamin K-dependent clottingzymogens comprising at least FII, FX and optionally FIX and/or FVII;

c) optionally providing the inhibitor of at least one of the vitaminK-dependent clotting zymogens;

d) admixing the components of a) and b) or a) to c).

In one embodiment, each of the components a) to c) is free of anirreversible thrombin inhibitor.

In some embodiments, at least one of the components is provided inliquid form, thereby resulting in a liquid formulation. In someembodiments, the formulation is dried.

In another aspect, provided is a method of providing hemostatictreatment; graft fixation, wound healing and/or anastomosis, to asurface in a subject, comprising applying to the surface a calcium-freesealant formulation comprising an effective amount of fibrinogen; andvitamin K-dependent clotting zymogens comprising at least FII, FX, andoptionally an inhibitor of at least one of the vitamin K-dependentclotting zymogens and/or Factor IX and/or Factor VII; wherein theformulation is free of an irreversible thrombin inhibitor.

The sealant formulation can be used for treating, without limitation,abdominoplasty; tissue healing; burn treatment; and dura sealing,hemostasis; graft fixation; wound healing; anastomosis. The surfacemaybe a bleeding or non-bleeding surface.

The invention provides a method of healing and/or reducing blood loss ina subject in need, comprising applying to the subject a therapeuticallyeffective amount of a formulation according to the invention.

The term “a therapeutically effective amount” refers to the doserequired to prevent, ameliorate, and/or treat a disease, disorder orcondition. The effective dose can be changed depending on the age andweight of the subject, the disease or condition, its severity and otherfactors which can be recognized by the skilled in the art.

In another aspect, provided is a calcium-free sealant formulationcomprising an effective amount of fibrinogen; and vitamin K-dependentclotting zymogens comprising at least FII, FX, and optionally areversible inhibitor of at least one of the vitamin K-dependent clottingzymogens and/or FIX; wherein the formulation is free of an irreversiblethrombin inhibitor for use in hemostasis; graft fixation; wound healing;anastomosis. The use includes, without limitation, abdominoplasty;tissue healing; burn treatment; and dura sealing,

The vitamin K-dependent clotting zymogens in the formulations disclosedherein may further comprise Factor IX and or Factor VII.

The formulations disclosed herein may further comprise Factor V.

The fibrinogen can be prepared from initial blood composition. The bloodcomposition can be whole blood or blood fractions, i.e. a product ofwhole blood such as plasma. Fibrinogen can be autologous, humanincluding pooled plasma, or of non-human source. It is also possiblethat the fibrinogen is prepared by recombinant methods or can bechemically modified.

In one embodiment of the invention, the fibrinogen solution is comprisedfrom a biologically active component (BAC) which is a solution ofproteins derived from blood plasma which can further comprise antifibrinolytic agents such as tranexamic acid and/or stabilizers such asarginine, lysine, their pharmaceutically acceptable salts, or mixturesthereof. BAC can be derived from cryoprecipitate, in particularconcentrated cryoprecipitate.

The term “cryoprecipitate” refers to a blood component which is obtainedfrom frozen plasma prepared from whole blood. A cryoprecipitate can beobtained when frozen plasma is thawed in the cold, typically at atemperature of 0-4° C., resulting in the formation of precipitate thatcontains fibrinogen and factor XIII. The precipitate can be collected,for example by centrifugation and dissolved in a suitable buffer such asa buffer containing 120 mM sodium chloride, 10 mM trisodium citrate, 120mM glycine, 95 mM arginine hydrochloride. The solution of BAC cancomprise additional factors such as for example factor VIII,fibronectin, von Willebrand factor (vWF), vitronectin, etc. for exampleas described in U.S. Pat. No. 6,121,232 and WO9833533. The compositionof BAC can comprise stabilizers such as tranexamic acid and argininehydrochloride. The amount of tranexamic acid in the solution of BAC canbe from about 80 to about 110 mg/ml.

In another embodiment, the concentration of plasminogen and plasmin inthe BAC composition is lowered to equal or less than 15 μg/ml like forexample 5 μg/ml or less plasminogen e.g. using a method as described inU.S. Pat. No. 7,125,569, EP 1,390,485 and WO02095019. In anotherembodiment of the invention, when the concentration of plasminogen andplasmin in the BAC composition is lowered, the composition does notcontain tranexamic acid or aprotinin.

The fibrinogen solution may be the BAC2 component (from EVICEL®) or anyother fibrinogen containing solution, such as purified recombinantfibrinogen or cryoprecipitate produced from human plasma.

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description of theinvention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows fibrin clotting of a PPSB/BAC2 (BAC2 as a source offibrinogen) liquid formulation after its application onto a piece ofcorium (derived from bovine hide) coated with tissue factor and calcium.Clot formation was determined by monitoring the gelation of theformulation, and the fact that the formulation ceased to flow. Formationof thrombin (left dish, arrows) was observed by coloration (achromogenic substrate for thrombin was added to the samples) of the clot30 seconds after application to the coated-corium. The Fig. shows theclot 3 minutes after the application. No clotting or color was observedwith a liquid solution comprising saline and BAC2 (right dish).

FIG. 2A is a graph showing the effect of adding an increasing percentageof PPSB prepared at 10× concentration (10 IU Factor II/ml) to BAC2(fibrinogen) on clotting time. FIG. 2B is a graph showing the effect ofchanging the ratio of Factor II and BAC2 on clotting time.

FIG. 3 shows clotting time of a formulation which includes PPSB preparedat 10× concentration (10 IU Factor II/ml) and BAC2 versus a formulationwhich includes plasma and BAC2 in different ratios.

FIG. 4A shows a PPSB-fibrinogen liquid formulation subjected to smallmolecule exchange by passing the formulation through a commercial columnpre-equilibrated with a buffer lacking CaCl₂. No clotting occurred evenafter several days.

FIG. 4B shows a PPSB-fibrinogen liquid formulation subjected to a smallmolecule exchange by passing the formulation through a commercial columnpre-equilibrated with a buffer including 40 mM CaCl₂. Following theexchange, a fibrin clot was spontaneously formed within 12-24 minutes.

FIG. 5 shows the effect of increasing calcium concentration in aPPSB-BAC2 formulation on clot formation rate (8-40 mM).

FIG. 6 shows the clotting time for PPSB:fibrinogen liquid formulationswith or without heparin stored at 4° C. or 23° C.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention relates to a sealant formulation comprisingfibrinogen; vitamin K-dependent clotting zymogens comprising at leastFactor II and Factor X; and a reversible inhibitor of at least one ofthe vitamin K-dependent clotting zymogens, wherein the formulation isfree of an added irreversible thrombin inhibitor.

The present invention is based, in part, upon the finding that a singleformulation comprising prothrombin zymogens (e.g. PPSB including FactorsII, IX, X, and VII) and fibrinogen (e.g. BAC2) is useful as a biologicalsealant, is stable and exhibits a long shelf life, as determined by thecapability of the formulation to form a clot e.g. after a prolongedstorage. Stability can be determined by observing minimal or absence ofspontaneous clotting in the formulation e.g. the formulation does notshow or have spontaneous clotting in the absence of an activator, suchas free calcium, and has an acceptable clotting activity level uponexposure to the activator.

It was found that storing a PPSB:fibrinogen formulation at 2-8° C. forup to 28 days or up to 90 days resulted in a stable formulation having afast clotting time upon CaCl₂ addition. Without wishing to be bound totheory, following storage at 2-8° C. a conversion of Factor X, VIIand/or IX zymogens to an active conformation may occur albeit theinhibition of arginine and citrate, thereby shortening the required timefor generation of active thrombin (after addition of calcium) andconsequently of clot formation. It was found that this fast clotting isprevented when the 2-8° C. stored formulation comprised 0.125 IU/mLheparin.

It was found that storing a PPSB:fibrinogen formulation at RT for up to28 days resulted in a stable formulation having a substantiallyunaltered clotting time upon CaCl₂ addition.

In one embodiment, the formulation comprises Factor X, VII and/or IX intheir active form.

It was found that fast clotting time was obtained in the presence ofboth, CaCl₂ and tissue factor.

In another aspect, the invention relates to a formulation free ofcalcium and includes fibrinogen and vitamin K-dependent clottingzymogens comprising at least Factor II and Factor X.

In some embodiments, the formulations further comprise factor IX and/orFactor VII.

The present invention is based, in part, upon the finding that a singleformulation comprising prothrombin zymogens and fibrinogen is useful asa biological sealant, is stable and exhibits a long shelf life, asdetermined by the capability of the formulation to form a clot.Stability can be determined by observing minimal or absence ofspontaneous clotting in the formulation e.g. the formulation does notshow or have spontaneous clotting in the absence of an activator, suchas free calcium, and has an acceptable clotting activity level uponexposure to the activator.

The terms “stable”, and “stability” when referring to a liquid mixture,mean substantially an absence of fibrin polymerization/clotting in theformulation before it contacts the activator and/or before thereversible inhibitor is removed, neutralized, blocked, and/or diluted.

The clotting activity level or capability of the formulation to form asealant can be determined in-vitro and/or in-vivo. Stability can also bedetermined by measuring and/or observing the presence of minimal orabsence of fibrin formation in the shelf-ready aqueous formulation.

Clotting can be measured, for example, by measuring migration length ona slanted surface (or drop test model) or by any other method known inthe art. Full clotting can be assessed by cessation of flow of theliquid formulation e.g. upon inversion. Rapid polymerization can bemeasured using a Stat4 clotting analyzer Stago Diagnostics or equivalentcoagulometer.

An acceptable clotting activity level means, for example, the ability ofthe formulation to form a clot within 30 minutes or less followingcalcium addition, and under 5 minutes following calcium and tissuefactor addition; and/or in-vivo, for example, within under 5 minutesfollowing calcium addition and/or contact with tissue factor (forexample, endogenous tissue factor).

In one embodiment the ability of the formulation to form a clot rangesbetween 30 seconds and 120 seconds on a bleeding surface containingtissue factor, and between 60 seconds and 600 seconds on a non-bleedingsurface.

Prothrombin, the inactive precursor of thrombin, does not displayproteolytic activity until the active form of the enzyme is generated byproteolytic cleavage of prothrombin by Factor Xa (activated Factor X).When desired, prothrombin can be activated to thrombin to convertfibrinogen to fibrin and attendant fibrin polymerization. Prothrombinand fibrinogen, in contrast to thrombin and fibrinogen, are stabletogether in solution.

In one embodiment in the formulation disclosed herein, prothrombin(Factor II) is included together with Factor X for its activation.Factor VII and factor IX are optionally included. Without wishing to bebound to theory, the presence of prothrombin, and the attendant absenceof thrombin, provides a formulation in which the kinetic conversion offibrinogen to fibrin may be well-controlled. The fibrin sealant may,therefore, be used in indications where the classic sealant isineffective, for example graft fixation. Endogenous activated zymogensmay facilitate conversion of prothrombin to thrombin at the wound site.

In one embodiment, a mixture of inactive enzyme precursors (also calledzymogens) is referred to as PPSB.

In some embodiments, a concentrate of the PPSB is a prothrombin complexconcentrate (PCC). The PCC can be a three-factor PCC (3F-PCC) with FII,FIX and FX, or a four-factor PCC (4F-PCC) which also includes FactorVII.

Disclosed herein is a fibrin sealant in which all the componentsrequired to form a fibrin are found in a single formulation which can beapplied from a single syringe, which improves ease of use andconvenience.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, steps or components but do not preclude the addition of one ormore additional features, steps, components or groups thereof.

When a numerical value is preceded by the term “about”, the term “about”is intended to indicate +/−10%.

“Thrombin” or “thrombin polypeptide” is a mammalian serine proteasewhich is part of the blood coagulation cascade and converts fibrinogeninto fibrin monomers which assembly into insoluble strands of fibrin, aswell as catalyzing other coagulation-related reactions. In humans,prothrombin is encoded by the F2 gene, and the resulting polypeptide isproteolytically cleaved in the coagulation cascade to form thrombin.Thrombin serves, inter alia, as an active component in severalhemostasis products. For example, fibrin sealants typically comprise afibrinogen component and a thrombin component. When both components aremixed (e.g. when applied to a bleeding wound) thrombin cleavesfibrinogen and a fibrin polymer is formed.

Thrombin is a serine protease which results from the cleavage ofprothrombin (Factor II), a zymogen precursor, by another serine protease(Factor Xa). Human thrombin is a 295 amino acid protein composed of twopolypeptide chains joined by a disulfide bond.

Various thrombin inhibitors are recognized in the art. An irreversiblethrombin inhibitor comprises a group of molecules that covalently bindthrombin or bind thrombin with a very high affinity and/or a group ofmolecules that destroy a functional group on thrombin or render thethrombin inactive. For example, hirudin and antithrombin III areconsidered herein as such irreversible thrombin inhibitors. Thrombinbinds to antithrombin III such that thrombin is not released from thecomplex. As used herein, a thrombin inhibitor that binds thrombin with ahigh affinity (sub-microM) is considered irreversible. One such exampleis hirudin, which binds thrombin in the picoM range.

In some embodiments, the formulation disclosed herein is free of addedthrombin, and is free of a thrombin inhibitor. The vitamin K-dependentclotting zymogens may be provided as, for example, PPSB or PCC. Anexample of a 20× fold PCC concentrate is Octaplex® (Octapharma, Vienna).Non-limiting examples of PCC include Beriplex®, Ocplex®, Kcentra®,Cofact®, among others.

For long-term storage, the formulation is aliquoted into sterile vials,ampoules, or other containers, for example a syringe or otherapplicator, which are then sealed. In one embodiment, a container thatpermits removal of the formulation with a syringe through the seal isused. The container is labeled according to standard practice in thepharmaceutical or medical device field. For use, the sealant formulationcan be used directly from the container according to the needs of theindividual patient and on the severity of bleeding. The formulation canbe applied to bleeding tissue to achieve hemostasis.

The liquid formulation disclosed herein is advantageous in that itremains stable for at least 14 days, 30 days, 45 days, 60 days or up to90 days, at an ambient temperature of about 2° C. to 8° C. or at least 7days at an ambient temperature in a range of about 2° C. and up to roomtemperature or for about 30 days at room temperature.

The formulation is assessed for stability by testing its capability toform a sealant when (i) removing, diluting, neutralizing and/or blockingthe inhibitor of at least one of the vitamin K-dependent clottingzymogens and/or (ii) adding a small molecule activator of the vitaminK-dependent clotting zymogens.

The formulation according to the present invention can be frozen orlyophilized.

Inter alia, the advantages of the present formulations are manifold andcan be at least one of the following: long shelf life, for example,stable as defined herein; good control of the kinetics of fibringeneration, for example, effectively no premature polymerization;purification of thrombin is not required, thereby reducing the costassociated with manufacturing; and/or easy to use and convenient toprepare; i.e. fewer components and no assembly required by attendingpractitioner.

The term a “pharmaceutically acceptable carrier” refers to any diluentor a vehicle which is suitable for human or other animal use. E.g. “apharmaceutically acceptable carrier or diluent” refers to reagents,compounds, materials, compositions, diluents that are compatible withthe constituents in the formulation and suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio. A pharmaceutically acceptable carriersuitable for use with the formulation disclosed herein includes liquids,semi-solid and solid materials.

A “surface” is a position or location where one desires to form thesealant or glue. The surface depends on the use of the sealant. Thesealant may be used, for example, in hemostasis, tissue fixation, graftfixation, wound healing and anastomosis. The formulations, methods, andkits disclosed herein can be used internally and externally, for tissueand organ graft fixation, for sealing a surgical wound, in vascularsurgery including providing hemostasis and for anastomoses such asarterial, gastrointestinal and tracheal anastomoses.

The surface can be an external surface of the skin that can be seen byunaided vision and a surface of an internal body part which is a part ofthe internal anatomy of an organism. External surfaces include, but arenot limited to, the skin of the face, throat, scalp, chest, back, ears,neck, hand, elbow, hip, knee, and other skin sites. Examples of internalbody parts include, but are not limited to, body cavity or anatomicalopening that are exposed to the external environment and internal organssuch as the nostrils; the lips; the ears; the genital area, includingthe uterus, vagina and ovaries; the lungs; the anus; the spleen; theliver; and the cardiac muscle. The surface can be a bleeding or anon-bleeding site. The surface can also be a working surface outside thebody.

A “subject” as used herein, includes animals of mammalian origin,including humans. In one embodiment, a subject is a surgery patient or awounded patient.

While the following examples demonstrate certain embodiments of theinvention, they are not to be interpreted as limiting the scope of theinvention, but rather as contributing to a complete description of theinvention.

EXAMPLES Example 1 Preparation of a Single Component Sealant FormulationComprising Vitamin K-Dependent Clotting Zymogens and Fibrinogen

PPSB, a source of the vitamin K-dependent clotting zymogens, wasstandardly produced as described in the art (Production of plasmaproteins for therapeutic use. Joseph Bertolini, Neil Goss, John Curling.2013 Wiley Press).

Briefly, concentrated PPSB, was produced by loading cryo-depleted humanplasma on a DEAE anion exchange column and eluting with a concentratedsalt solution (0.25M NaCl) which also includes 10 mM sodium citrate(NaCitrate). The PPSB was concentrated between 4-16 fold vs. plasma asdetermined by the prothrombin concentration (Factor II).

The mixture comprised all of the vitamin K-dependent clotting zymogensthat typically bind to anion exchange columns (such as FVII, FIX,protein C and protein S, and FX), their associated co-zymogens (FV andFVIII) and any other proteins that are co-eluted.

The vitamin K-dependent clotting zymogens inhibitors (e.g. NaCitrate,EDTA) served to chelate calcium ions and prevent premature activation ofany of the prothrombin complex comprising FII, FV, and FX, or any otherCa²⁺ dependent process such as the Tenase complex activation (FVIII andFIX) or FXIII activation.

A 10-fold PPSB concentrate was added to a fibrinogen solution, themixture comprised between 3-4% concentration of clottable protein (7%fibrinogen diluted 1:1 with PPSB). The fibrinogen solution used in theExamples was BAC2 (a fibrinogen comprising component from EVICEL® FibrinSealant). Final ratio between FIX and fibrinogen was 0.14 units/mg (i.e.0.14 Units (U) FIX per mg fibrinogen). This mixture was shown to bestable at 2°-8° C. for at least three months without showing anypremature fibrin clot formation.

Example 2 Clot Formation Using the Single Component Sealant Formulation

To assess the ability of the single component sealant formulation toform a clot, a simulated bleeding site was created by using a piece ofcorium (bovine hide) coated with a layer of PT (Prothrombin Time)reagent containing tissue factor (Diagnostica Stago STA Neoplastin CIPlus, Cat #00606) and calcium, at about 15-25 mM.

A liquid solution comprising equal volumes of PPSB and BAC2 (as preparedin Example 1) was applied to the tissue factor coated corium and therate of clot formation was assessed by, the PT assay, a test thatmeasures how long it takes to form a clot, typically, after addition oftissue factor and calcium.

The arginine present in BAC2 has an inhibitory effect on thrombinactivity, however, it is not sufficient to completely inhibit orinactivate the thrombin which is generated (from prothrombin) via thetissue factor pathway. Typically, NaCitrate, present in the BAC2 andPPSB, inhibits generation of active enzymes by chelating calcium.However, the PT reagent contains calcium at a level sufficient toovercome the NaCitrate inhibitory effect.

Clotting was initially observed ˜30 seconds after applying the liquidsolution onto the coated corium and the clot was fully solid after about1 minute and 30 seconds. As a control, a solution comprising equalvolumes of BAC2 and saline was applied to the coated-corium and assessedfor clot formation. In the absence of PPSB, no clot was formed duringthe observation period (more than 30 minutes).

To further evaluate the enzymatic properties of the liquid sealantformulation (PPSB/BAC2), a chromogenic thrombin substrate[H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline dihydrochloride.S-2238™ Chromogenix] was added to the two samples (PPSB/BAC2 andBAC2/saline). The PPSB/BAC2 sample (left dish in FIG. 1) turned yellowdue to thrombin generation and cleavage of the chromogenic substrate.However, the BAC2/saline sample (right dish) did not turn yellow,indicating that thrombin was not generated in the sample.

FIG. 1 shows coloration of the PPSB/BAC2 clot after incubation of threeminutes (left). This coloration was already evident 30 seconds after theapplication. No color was observed with the BAC2/saline sample (right).

Example 3 Effect of Zymogens (PPSB) to Fibrinogen Ratio on Clotting Time

In this experiment, BAC2 having a fibrinogen concentration of 70 mg/mLand PPSB, both as described above were used. The amount of Factor IX andprothrombin (Factor II) present in the PPSB was approximately 10-foldmore than the amount present in plasma, i.e. about 9.8 IU/ml Factor IXand 10 IU/mL Factor II. PPSB and BAC2 were mixed in different ratios,see FIG. 2A, and the clotting time was measured by the PT assay using acoagulation analyzer (Diagnostica Stago Start4). Clotting was induced byadding the Neoplastin PT reagent which comprises, both calcium andtissue factor which bind to Factor VII thereby initiating the extrinsicpathway of coagulation. 100 μL Neoplastin PT reagent (kept well mixed at37° C.) was combined with 500 μL of test sample (the BAC2 and PPSB mixat the different volume ratios e.g. 90:10, 80:20, 70:30 and so on,respectively). The test sample was incubated at 37° C. for 60 secondsprior the assay in the incubation wells of the analyzer. Theconcentration of fibrinogen was variable depending on the volume ratioof BAC2 and PPSB, however, in any case the fibrinogen was present in thesample in excess (even if diluted 20:1 with PP SB), and therefore,without being bound to theory, the rate limiting factor was the rate ofenzyme activation.

FIGS. 2A.-2B and Table 1 show the clotting time of the differentformulations. FIG. 2A shows clotting as a function of PPSB percent inBAC2, and FIG. 2B and Table 1 shows clotting as a function of the ratioof Factor II to fibrinogen (U per mg). Typically, Factor II is used toexpress the activity of PPSB in commercially available products. Theamount of Factor IX and prothrombin present in the PPSB is approximately10 fold more than the amount present in plasma, i.e. 10 IU/mL Factor II.

TABLE 1 Clotting time (in sec) of PPSB:BAC2 solution having differentFII to Fibrinogen ratios (Units per mg). Clotting FII to Time Fibrinogensec U/mg 15.0 3.26 13.7 1.54 15.1 0.69 17.6 0.17 30.9 0.04 45.2 0.0275.0 0.01

The results in FIG. 2A and Table 1 show that increasing PPSB volume (asnormalized by Factor II) compared to the fibrinogen (BAC2) volumedecreased the clotting time of the mixture. At 50% PPSB in BAC theclotting time was about 17.5 sec, and decreased slightly with higheramounts of PPSB.

Example 4 Effect of Substituting Plasma for PPSB

The present experiment examines the effect of using plasma instead ofPPSB in the formulation.

Formulations comprising plasma/BAC2 and PPSB/BAC2 were prepared at thefollowing volume ratios: 1-50% PP SB+50% BAC2; 2-20% PPSB+80% BAC2;3-20% plasma+80% BAC2; and 4-50% plasma+50% BAC2. PPSB was prepared asin Example 1 (10× concentrated FIX, FII compared to plasma),

As in Example 3 above, clotting was induced with tissue factor (TF) andcalcium.

The results in FIG. 3 show that using equal volume percentageformulations (1-4 and 2-3), a faster clotting time was obtained with thePPSB/BAC2 formulation (1 and 2) as compared to the plasma/BAC2formulation (3 and 4, respectively).

These results demonstrate that PPSB, which is enriched in vitaminK-dependent clotting zymogens, and fibrinogen can be used to accelerateclot formation.

Example 5 Fibrin Clot Formation Using a Small Molecule Exchange Column

A formulation comprising PPSB and BAC2 was prepared as follows: PPSB wasprepared as in Example 1 and mixed at a one to one volumetric ratio withBAC2, finally yielding approximately 3.5% (w/v) clottable protein [theinitial BAC2 contains 70 mg/ml (or 7% clottable protein, mostlyfibrinogen)] and a PPSB that was concentrated approximately 5-fold ascompared to plasma (approximately 0.14 international units (IU) ofFactor II per mg of fibrinogen). The formulation also included 1-2 mM ofEDTA, 10 mM NaCitrate, 1% (w/v) arginine*HCl, and glycine and acetatebuffer (pH 7.0; the buffer comprised 1% (w/v) glycine and 20 mMacetate).

The formulation was passed through a commercially available bufferexchange spin column (Disposable PD-10 Desalting Columns, Product code:17-0851-01, GE Healthcare) either pre-equilibrated with CaCl₂ solution(40-50 mM) or with water, 1% (w/v) glycine buffer containing 2% (w/v)arginine (but no CaCl₂) and flow through formulation was collected in atube.

Clotting was assessed by inverting the tube containing thebuffer-exchanged formulation.

The results show that a formulation which passed through the columnpre-equilibrated with CaCl2 clotted spontaneously (FIG. 4B) within lessthan 30 minutes, whereas the formulation which passed through the bufferlacking CaCl2 did not clot, even after several days (FIG. 4A).

Example 6 The Effect of CaCl₂ Concentration on the Rate of Fibrin ClotFormation

The following example explores the effect of adding different CaCl₂concentration to the single component sealant formulation on the rate ofclot formation.

A 10-fold enriched PPSB10 IU FII/ml):BAC2 formulation (1:1 prepared asin example 1, and without EDTA) was mixed with a PT reagent (a mixtureof tissue factor and phospholipids and lacking calcium). CaCl₂ atincreasing concentrations was supplemented to the mix and the clottingtime was measured.

Results are seen in FIG. 5. Rapid clotting (<20 seconds as measuredusing a Stat4 clotting analyzer Stago Diagnostics) was observed at about8 mM to about 30 mM calcium.

Example 7 Effect of CaCl₂ on Clot Formation

It has been found that a clot was formed upon addition of calcium to thestable formulation. In this experiment, PT reagent was not added (i.e.no tissue factor or phospholipids were added). Experiments with 10-foldenriched PPSB (as compared to plasma) mixed 1:1 with BAC2 showed thatclotting time could be achieved between 5-10 minutes by adding 10 mMcalcium in the absence of tissue factor.

Example 8 Assessment of Stability of PPSB:BAC2 Formulation at 2-8° C.

In this example, the stability of the single component fibrin sealantformulation containing PPSB and fibrinogen was evaluated at atemperature of 2-8° C.

For this purpose, a PPSB-BAC2 formulation (ratio of 0.14 U FII per mgfibrinogen) was incubated at 2-8° C. for different time points (up to 90days), and the formulation was tested for its ability to form a clotwithin 30 minutes.

Clot formation was initiated by subjecting the formulation to a PD-10pre-packed column (Sephadex™ G-25, GE Healthcare 17-0851-01). The columnwas equilibrated with 5 ml buffer containing 50 mM CaCl₂ (Sigma) and 20mM NaAcetate pH 7.00 (Sigma) three times in gravity mode. An additional5 ml of CaCl₂ were applied to the column and the column was centrifugedfor 2 min at 1000 g at 20° C. The column was used to completely removesmall molecule inhibitors including EDTA which was present in theformulation at a concentration of 2.5 mM.

The pre-incubated PPSB-BAC2 formulation was warmed for 10 minutes in a37° C. water bath, applied to the column, the column was centrifuged for2 min at 1000 g at 20° C. (see Table 2 for the formulation volumeapplied to the column) and the column flow through solution wascollected.

Following collection, time to clot initiation/gelation was assessed inthe collected material by visually observing a change in the coloration(from clear to opaque). Also, time to complete clotting was assessed bycessation of flow of the collected material upon inversion.

Results of clot initiation and complete clotting time are presented inTable 2, below.

TABLE 2 Clot initiation and complete clotting time of formulationincubated at 2-8° C. for different periods of time. Volume Time to clotTime to Time of mix initiation/ complete point applied gelation clotting(days) (ml) (min) (min) 0 2.5 22 25 3 2.48 19 23.5 7 2.45 15 21 14 2.514.5 19 30 2.5 13 16.5 60 2.45 7.5 17 90 2.4 6 11.5

The time to complete clotting was at all times less than or equal to 25minutes. Some shortening of the required time occurs, without wishing tobe bound to theory, by the slow conversion of a small amount of the PPSBzymogens to an active conformation, albeit inhibited by arginine andcitrate, thus shortening the required time for the generation of activethrombin. The results indicate that the formulation is stable for atleast up to 90 days at a temperature of 2-8° C.

Example 9 Animal Model for In-Vivo Assessment of the Formulation

The rat kidney hemostasis model is a common model to test the ability ofa tested formulation to achieve hemostasis (Raccuia J S et al., Am JSurg. 1992. 163(2):234-8. Comparative efficacy of topical hemostaticagents in a rat kidney model).

Briefly, the kidney was dissected out of the side of the peritoneum andpads were placed around it to soak up any bleeding. A clamp was placedon the blood vessels supplying the kidney and a traverse cut was madethrough the kidney. The tested formulation was applied and the clamp wasremoved. Bleeding was assessed over a one hour period, after which thetotal amount of bleeding was weighed. Subsequently, the formulation wasscraped off and the bleeding allowed to resume and quantified as low,medium, or high (to assess that the bleeding potential was stillexisting). All rats were infused with 300 IU heparin/kg animal weight tomake the bleeding model more challenging.

One albino rat weighing 406 grams (g) was anaesthetized and subjected tothe rat kidney hemostasis model using the PPSB:BAC2 formulation with afinal concentration of 5 IU/ml PPSB: 3.5% fibrinogen. A classic2-component commercial fibrin sealant was used as reference.

Results:

A PPSB:BAC2 formulation was mixed with CaCl₂ (25 mM final concentrationin the formulation; CaCl₂ was manually added into the formulation) andincubated for 15 minutes at room temperature before application to thekidney surface. A clot was formed on the surface immediately uponapplication and bleeding was completely stopped after 38 minutes. Thetotal blood loss was 5.9 g over the one hour period of the model.

Using a classic 2-component commercial fibrin sealant resulted inimmediate clot formation and a total blood loss in the range of 0-10.3 g(in 15 animals). The commercial fibrin sealant was applied directly ontothe kidney surface without incubation.

Thus, the all in one PPSB-based fibrin sealant formulation disclosedherein has a good hemostatic potential.

Example 10 Assessment of Stability of PPSB:BAC2 Formulation at RT and2-8° C. With or without Heparin

In this example, the stability of a single component fibrin sealantformulation containing PPSB and fibrinogen was evaluated.

The PPSB was prepared as described in Example 1. BAC2 (BiologicallyActive Component 2) was used as the fibrinogen component, whichcontained approximately 100 mg/mL total protein including 70 mg/mLclottable fibrinogen, 20 mg/mL arginine, 10 mM sodium citrate, andexcipients including glycine and sodium chloride). The PPSB was combinedwith BAC2 in equal volumes to generate the single component fibrinsealant formulation.

The samples from the single component fibrin sealant formulation werealiquoted and stored either at room temperature (20-25° C.) or in therefrigerator (2-8° C.).

To a second set of samples, unfractionated heparin at 0.25 IU/mL wasadded to the PPSB, resulting in a final heparin concentration of 0.125IU/mL in the single component fibrin sealant formulation.

Stability was assessed using a standard prothrombin time (PT) assay atvarious time points over a 28 day period. To perform the PT assay, 50 μLof the sample warmed to 37° C. was combined with 100 μL of PT reagent(Diagnostica Stago STA Neoplastin CI Plus), which consisted of tissuefactor and 10 mM calcium. A Diagnostica Stago STart4 coagulationanalyzer was used to determine the rate of clot formation. The resultsare shown in FIG. 6. The results show that at 6 days, clotting times forall of the formulations stored at either temperature with/withoutheparin were comparable to the baseline clotting time values. Therefrigerated formulation without heparin showed a reduction in clottingtimes at 18 days, and at 28 days the sample was gelled due to activationof clotting factors. No trends in clotting times were observed duringthe 28 day stability study for the formulation with added heparin storedin the refrigerator or the formulations stored at room temperature.

Although various embodiments have been described herein, manymodifications and variations to those embodiments may be implemented.Also, where materials are disclosed for certain components, othermaterials may be used. The foregoing description and following claimsare intended to cover all such modification and variations.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialsdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

1. A sealant formulation comprising fibrinogen; vitamin K-dependentclotting zymogens comprising at least Factor II and Factor X; and atleast one reversible inhibitor of at least one of the vitaminK-dependent clotting zymogens, wherein the formulation is free of anadded irreversible thrombin inhibitor.
 2. The formulation of claim 1,wherein the formulation is in liquid form and comprises apharmaceutically acceptable carrier.
 3. The formulation of claim 1,wherein the formulation further comprises Factor V.
 4. The formulationof claim 1, wherein the vitamin K-dependent clotting zymogens furthercomprise Factor VII.
 5. The formulation of claim 4, wherein the vitaminK-dependent clotting zymogens further comprise Factor IX.
 6. Theformulation of claim 5, wherein Factor X, Factor VII, and/or Factor IXare, at least partially, in their active form.
 7. The formulation ofclaim 1, wherein the reversible inhibitor is selected from the groupconsisting of heparin, a calcium chelator, a serine protease active siteinhibitor and a combination thereof.
 8. The formulation of claim 7,wherein the formulation is in liquid form and is stable for at least 14days at an ambient temperature of about 2° C. to 8° C.
 9. Theformulation of claim 2, wherein the liquid formulation remains stable atleast 7 days at an ambient temperature of about 2° C. and up to roomtemperature.
 10. The formulation of claim 9, wherein the liquidformulation is stable for about 30 days at room temperature.
 11. Theformulation of claim 1, being free of added thrombin.
 12. Theformulation of claim 1, wherein the irreversible thrombin inhibitor ishirudin.
 13. The formulation of claim 1, wherein the irreversiblethrombin inhibitor is anti-thrombin III.
 14. The formulation of claim 1,wherein the vitamin K-dependent clotting zymogens is provided as aconcentrate, concentrated by about 2-50 fold compared to theirconcentration in plasma, as normalized to Factor II.
 15. The formulationof claim 14, wherein the vitamin K-dependent clotting zymogenconcentrate is Prothrombin Complex Concentrate (PCC).
 16. Theformulation of claim 1, wherein the ratio of the vitamin K-dependentclotting zymogens (U) to fibrinogen (mg clottable protein) is about 0.01to about 1.0, as normalized to Factor II.
 17. The formulation of claim7, wherein the reversible inhibitor is a calcium chelator.
 18. Theformulation of claim 17, wherein the calcium chelator is selected fromthe group consisting of a citrate ion, oxalate, EDTA, EGTA and acombination of such calcium chelators.
 19. The formulation of claim 18,wherein the calcium chelator is a citrate ion.
 20. The formulation ofclaim 19, wherein the citrate ion is provided by sodium citrate.
 21. Theformulation of claim 20, comprising from about 1 mM to about 50 mMsodium citrate.
 22. The formulation of claim 21, comprising from about 5mM to about 25 mM sodium citrate.
 23. The formulation of claim 18,comprising from about 0.1 mM to about 2.5 mM EDTA and/or EGTA.
 24. Theformulation of claim 7, wherein the reversible inhibitor is a serineprotease active site inhibitor.
 25. The formulation of claim 24, whereinthe reversible inhibitor is arginine.
 26. The formulation of claim 25,comprising from about 0.1% to about 5% (w/v) arginine.
 27. Theformulation of claim 1, for use in hemostasis, healing and/or surgery.28. A method for preparing a sealant at a surface comprising: providingthe formulation of claim 1; and applying the formulation to the surfaceunder conditions which facilitate fibrin polymerization at the surface.29. The method of claim 28, wherein the surface is a bleeding ornon-bleeding surface in a subject.
 30. The method of claim 28, whereinthe conditions comprise (i) removing, neutralizing, blocking and/ordiluting the reversible inhibitor and/or (ii) adding a small moleculeactivator of at least one of the vitamin K-dependent clotting zymogens.31. The method of claim 30, wherein the small molecule activator is adivalent cation.
 32. The method of claim 31, wherein the divalent cationis a calcium cation.
 33. The method of claim 32, wherein the calciumcation is provided by CaCl₂.
 34. A container comprising the formulationof claim
 1. 35. A kit comprising the container of claim 34, andoptionally instructions for use.
 36. A method of manufacturing a sealantformulation, comprising the steps of: a) providing a fibrinogencomponent; b) providing a component comprising vitamin K-dependentclotting zymogens including at least Factor II and Factor X; c)providing a component comprising at least one reversible inhibitor of atleast one of the vitamin K-dependent clotting zymogents; and d) admixingthe components a) to c); wherein the admixed components are free ofadded irreversible thrombin inhibitor.
 37. The method of claim 36,further admixing Factor V.
 38. The method of claim 36, wherein thevitamin K-dependent clotting zymogens further comprise Factor VII. 39.The method of claim 36, wherein the vitamin K-dependent clottingzymogens further comprise Factor IX.
 40. The method of claim 36, whereinthe fibrinogen and the reversible inhibitor are provided in the samecomponent.
 41. The method of claim 36, wherein at least one of thecomponents is provided in liquid form, thereby resulting in a liquidformulation.
 42. A formulation obtainable according to claim
 36. 43. Amethod of healing and/or reducing blood loss in a subject in need,comprising applying to the subject a therapeutically effective amount ofa formulation according to claim 1 or
 42. 44. A sealant formulationcomprising fibrinogen; vitamin K-dependent clotting zymogens comprisingat least Factor II, Factor IX and Factor X; and at least one reversibleinhibitor of at least one of the vitamin K-dependent clotting zymogen,wherein the formulation is free of added irreversible thrombininhibitor.
 45. A calcium-free sealant formulation comprising fibrinogenand vitamin K-dependent clotting zymogens comprising at least Factor IIand Factor X.
 46. A method for preparing a sealant at a surfacecomprising: providing the formulation of claim 44 or 45; and applyingthe formulation to the surface under conditions which facilitate fibrinpolymerization at the surface.
 47. The method of claim 46, wherein theconditions comprise adding a calcium cation.
 48. A container comprisingthe formulation of claim 44 or
 45. 49. A kit comprising the container ofclaim 48; and optionally instructions for use.
 50. A method ofmanufacturing a calcium-free sealant formulation, comprising a)providing a fibrinogen component; b) providing a component comprisingvitamin K-dependent clotting zymogens including at least Factor II andFactor X; and c) admixing the components of a) and b).